Supplementary Figure 3: GSC death occurs independent of AMPK-regulated mTOR and autophagy pathways.

a.WB of pULK1 (AMPK target) and autophagy substrate LC3 in AMPK-silenced GSCs. ULK1 and actin are used as loading controls. b, WB of autophagy substrates LC3 and P62 in NT or AMPKβ1shRNA expressing cells treated with Bafilomycin A showing autophagy flux. c, Viability of GSCs and HepG2 liver cancer line treated with autophagy inhibitors Bafilomycin and Chloroquine, or infected with NT or ATG13 shRNAs. (Average of two independent experiments). d, Superoxide detection by flow cytometry in NT or AMPKβ1 shRNA expressing GSCs using MitoSox Red. (Average of two independent experiments). e, Viability of GSCs expressing NT or AMPKβ1 shRNA treated with the reducing agent N-acetylcysteine (NAC) or the ACC inhibitor TOFA. (Average of two independent experiments). f, ATP levels in GSC lines expressing AMPKβ1 shRNA or NT shRNA at indicated times. ATP value was normalized to protein. (n = 3). *p ≤ 0.002; ⁺ ≤ 0.004. g, Viability of GSCs expressing NT or AMPKβ1 shRNA treated with the mTORC1 inhibitor rapamycin (5nM). (n = 3). *p ≤ 0.01; ⁺ ≤ 0.004. h, WB of stem cell markers Musashi and Nanog in GSCs expressing NT and AMPKβ1 shRNA. Error bars; mean +/- S/d. Statistical significance in above experiments was assessed using two-tailed t-test. n values represent independent experiments. Source data are available in Supplementary Table 4. All western blots represent data from 2-3 independent repeats. Unprocessed blots are shown in Supplementary Fig. 9.